Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Ma, Li; Bindokas, Vytautas P.; Kuznetsov, Andrey V.; Rhodes, Christopher J.; Hays, Lori B.; Edwardson, J. Michael; Ueda, Kazuya; Steiner, Donald F.; Philipson, Louis H.

doi:10.1073/pnas.0403201101

Cited by 89 publications

(101 citation statements)

References 27 publications

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“…Various studies have suggested that insulin granules undergo complete fusion resulting in the total mixing of granule membrane proteins and lipids with the plasma membrane (12)(13)(14). Alternatively, other studies have suggested that the insulin granules transiently open a fusion pore to allow intraluminal contents to diffuse out followed by membrane closure.…”

Section: Discussionmentioning

confidence: 99%

“…Initial studies have suggested that the release of the insulin-containing dense core granule content occurs en masse, consistent with the formation of a plasma membrane pore that fully expands to encompass the granule membrane proteins and lipids (12)(13)(14). Alternatively, it has also been suggested that insulin granules may actually stack and communicate with each other with a given granule releasing its content into another granule leading to a continuously gating channel to the plasma membrane through a process called compound exocytosis (15,16).…”

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confidence: 99%

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Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

Min

Leung

Tomás³

et al. 2007

Journal of Biological Chemistry

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The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 ␤-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/ K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of ␤-cell insulin secretion.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

Min

Leung

Tomás³

et al. 2007

Journal of Biological Chemistry

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“…Importantly, this distinction between "full" but reversible events versus complete merger is difficult, if not impossible to detect, unless the fates of membrane and cargo markers are imaged simultaneously [81,109]. This limitation may explain why recordings relying solely on soluble probes [112,113] tend to invoke complete vesicle merger as the principal mechanism of insulin release.…”

Section: Mechanisms Of Vesicle Release At the Cell Surface: Full Fusimentioning

confidence: 99%

Visualising insulin secretion. The Minkowski Lecture 2004

Rutter

2004

Diabetologia

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Insulin secretion from pancreatic islet beta cells is a tightly regulated process, under the close control of blood glucose concentrations, neural inputs and circulating hormones. Defects in glucose-triggered insulin secretion, possibly exacerbated by a decrease in beta cell mass, are ultimately responsible for the development of type 2 diabetes. A full understanding of the mechanisms by which glucose and other nutrients trigger insulin secretion will probably be essential to allow for the development of new therapies of type 2 diabetes and for the derivation of "artificial" beta cells from embryonic stem cells as a treatment for type 1 diabetes. I focus here on recent developments in our understanding of beta cell glucose sensing, achieved in part through the development of recombinant targeted probes (luciferase, green fluorescent protein) that allow islet beta cell metabolism and Ca 2+ handling to be imaged in situ in the intact islet with single cell resolution. Combined with classical biochemistry, these techniques show that the beta cell is uniquely poised, thanks to the expression of low levels of lactate dehydrogenase and plasma membrane lactate/monocarboxylate transporters, to channel glucose carbons towards oxidative metabolism, ATP synthesis and inhibition of AMP-activated protein kinase, a newly defined regulator of insulin release. I also discuss the molecular basis of the recruitment of secretory vesicles to the cell surface, analysed by the use of new imaging techniques including total internal reflection of fluorescence, as well as the "nanomechanics" of the exocytotic event itself.

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“…Cells were then harvested and purified using CsCl gradient ultracentrifugation (50), and viral titers were measured by the formation of viral plaques in sequential dilutions. Replicationdeficient Ad constructs were used: Ad-syncollin-GFP (kindly provided by Dr. Christopher Rhodes, University of Chicago) (20,27) and Ad-GFP (53). Mouse Rab27 sequences, fused to epitope tags on their NH 2 termini, were expressed using the following constructs: Ad-Xpress-Rab27bQ78L (constitutively active; Xp-CA), Ad-Xpress-Rab27bN133I (dominant negative; Xp-DN) and AdXpress-Rab27b (wild-type; Xp-WT), which were kind gifts of Dr. John Williams, University of Michigan (6,56); and Ad-YFPRab27b (YFP-WT), Ad-YFP-Rab27bQ78L (YFP-CA), Ad-YFPRab27bN133I (YFP-DN) as described (43).…”

Section: Methodsmentioning

confidence: 99%

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Chiang¹,

Ngo²,

Schechter

et al. 2011

American Journal of Physiology-Cell Physiology

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mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b Ϫ/Ϫ and Rab27 ash/ash /Rab27b Ϫ/Ϫ mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosomelike organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release. actin; Rab3d; exocrine secretion; syncollin; mouse models A FUNCTIONING LACRIMAL GLAND (LG) is critical for a healthy ocular surface. This exocrine gland is the primary source of tear proteins and fluid, which, released up on the gland's exposure to sympathetic or parasympathetic agonists provided by innervating nerves, contribute to the middle aqueous layer of the precorneal film. Much of the LG's secretions originate from acinar cells, which constitute over 80% of the mass of the LG (13). These LG acinar cells produce a diverse array of secretory proteins that are internally sorted into large pools of serous and mucous secretory vesicles (SV) sized ϳ1 m in diameter and stored beneath the apical plasma membrane (APM) in preparation for regulated exocytosis. SV contents include nutrients and growth factors (lacritin and EGF), antibacterial and antiviral factors (secretory IgA and lactoferrin), and an array of proteases and lysosomal hydrolases (10, 39, 47). Despite its physiological importance, however, few studies have focused on the mechanisms of secretory membrane trafficking in LG acinar cells, in part due to the fragility and heterogeneity of these LG acinar SV relative to those in other acinar secretory cells, e.g., pancreatic acini (7).The current schematic of exocytosis in the LG acinar cell suggests multiple participants. Mature LG acinar cell SV localize beneath an actin-rich network und...

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Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Cited by 89 publications

References 27 publications

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

Visualising insulin secretion. The Minkowski Lecture 2004

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

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